JP7105429B2 - Method and apparatus for inducing a hibernation-like state - Google Patents

Description

The present invention provides a method and apparatus for inducing a hibernation-like state.

Warmotherms, birds, and mammals expend most of their body energy for heat production in order to maintain internal body temperature (T _B ) within a narrow range above ambient temperature (T _A ). However, some mammals actively cool down to survive winter food shortages, a state known as hibernation. Animals return to normal without obvious tissue damage ^1,2 . Mice do not hibernate, but exhibit a short-term hypometabolic state known as diurnal diapause when they can benefit from a reduced basal metabolic rate. The reduction in energy expenditure in both hibernation and diurnal dormancy is primarily achieved by lowering body temperature, which is influenced by two major factors: the theoretical setpoint temperature (TR) and the negative feedback gain of heat production (H). be. In diapausing mice, TR remains close to normal, but H decreases to nearly 10-fold normal, resulting in a TB much lower than TR ³ . In contrast, both TR and H are significantly reduced in hibernation, allowing maintenance of a hypometabolic state more efficient than day-to-day hibernation and adaptable to fluctuations in ambient temperature ^4,5 . A number of experiments have established that this aggressive metabolic depression is regulated by the central nervous system ⁶ . However, its neural mechanism remains completely unknown. Elucidation of the mechanisms of daily dormancy and/or hibernation is a necessary step to develop methods for artificially inducing a hibernation-like hypometabolic state in non-hibernating animals, including humans ^1,7 , as well. will also be useful in long-range space exploration in the future. Here we find that excitatory manipulation of a novel, chemically defined neuronal population in the hypothalamus results in a very prolonged hypometabolic/hypothermic state in mice. In this state, the metabolic rate is reduced by more than a third, but unlike the anesthetized state, mice still respond to changes in ambient temperature. Moreover, mice spontaneously recovered from this condition without any apparent abnormalities. This discovery is an important finding for the development of hibernation mechanisms and methods for inducing an artificial hibernation-like state.

The present invention provides a method and apparatus for inducing a hibernation-like state.

In the brain of living, non-hibernating animal subjects, we have demonstrated that the hypothalamic anteroventral periventricular nucleus (AVPe), medial preoptic area (MPA) and periventricular nucleus (Pe) of the hypothalamus contain We have found that a hibernation-like state can be induced in the subject by applying an excitatory stimulus to pyroglutaminated RF amide peptide (QRFP) gene-expressing neurons within the region.

According to the present invention, for example, the following inventions are provided.
(1) in the brain of a living subject, within one or more regions selected from the group consisting of the anterior ventricular periventricular nucleus (AVPe), the medial preoptic area (MPA) and the periventricular nucleus (Pe); A device for stimulating pyroglutaminated RF amide peptide (QRFP)-producing neurons, comprising:
a control unit that transmits a control signal for controlling voltage generation;
a voltage generator that receives a control signal from the controller and generates a voltage;
a stimulation probe electrically connected proximally to said voltage generator and having an electrical stimulation electrode distally, said voltage generator having a length sufficient to access QRFP-producing neurons from the brain surface; a stimulation probe that generates electrical stimulation at a distal electrical stimulation electrode with a voltage from the body;
outside temperature gauge and
a core thermometer and
an exhaled gas analyzer for measuring oxygen concentration in exhaled gas;
a recording unit that records the measured outside temperature and at least one numerical value selected from the group consisting of core body temperature and oxygen concentration;
apparatus, including
(2) in the brain of a living subject, within one or more regions selected from the group consisting of the anterior ventricular periventricular nucleus (AVPe), the medial preoptic area (MPA) and the periventricular nucleus (Pe); A device for stimulating pyroglutaminated RF amide peptide (QRFP)-producing neurons, comprising:
a control unit that transmits a control signal that controls the release of the QRFP-producing neuron-stimulating compound;
a reservoir of said compound;
a compound delivery unit that receives a control signal from the control unit and delivers the compound from a compound storage unit;
a guide comprising a compound outlet and a channel for the compound to the outlet and delivering the compound to QRFP-producing neurons;
outside temperature gauge and
a core thermometer and
an exhaled gas analyzer for measuring oxygen concentration in exhaled gas;
a recording unit that records the measured outside temperature and at least one numerical value selected from the group consisting of core body temperature and oxygen concentration;
apparatus, including
(3) The apparatus according to (1) or (2) above, further comprising a determination unit that determines whether the subject is hypothermic based on the outside air temperature and core body temperature recorded in the recording unit.
(4) The above (1) to (3), further comprising a determination unit that determines whether the subject is in a hypometabolism state from the outside temperature, core body temperature, and oxygen concentration recorded in the recording unit. A device according to any one of the preceding claims.
(5) The above (1) to (4), further comprising a determination unit that determines whether the subject is in a hibernation-like state from the outside temperature, core body temperature, and oxygen concentration recorded in the recording unit. A device according to any of the preceding claims.
(6) GRFP continuously or intermittently until the control unit determines that the subject is in any one state selected from the group consisting of a hypothermic state, a hypometabolic state, and a hibernation-like state The device according to any one of (3) to (5) above, which transmits a control signal for stimulating a production neuron.
(7) A method of lowering the theoretical set point of body temperature in a mammalian subject, comprising providing an excitatory stimulus to pyroglutaminated RF amide peptide (QRFP)-producing neurons.
(8) the QRFP-producing neurons are neurons in one or more regions selected from the group consisting of the anterior ventral periventricular nucleus (AVPe), the medial preoptic area (MPA), and the periventricular nucleus (Pe); The method according to (7) above.
(9) The method according to (7) or (8) above, wherein the excitatory stimulus is a stimulus selected from the group consisting of chemical stimulation, magnetic stimulation and electrical stimulation.
(10) Excitatory stimulation of pyroglutaminated RF amide peptide (QRFP)-producing neurons present in the regions of the anterior ventral periventricular nucleus (AVPe), medial preoptic area (MPA), and periventricular nucleus (Pe) A method of screening for a substance that provides
contacting the test compound with the QRFP-producing neurons;
measuring the excitation of the QRFP-producing neurons;
selecting a test compound that provides an excitatory stimulus to the QRFP-producing neurons;
A method, including
(10a) specifically to pyroglutaminated RF amide peptide (QRFP)-producing neurons located within the regions of the anterior ventral periventricular nucleus (AVPe), medial preoptic area (MPA) and periventricular nucleus (Pe) A method for screening a substance that provides an excitatory stimulus, comprising:
causing the cell to express a receptor specifically expressed in QRFP-producing neurons;
contacting the test compound with the cell;
measuring the excitation of the QRFP-producing neurons;
selecting a test compound that provides an excitatory stimulus to the QRFP-producing neurons;
A method, including
(10b) specifically to pyroglutaminated RF amide peptide (QRFP)-producing neurons located within the regions of the anterior ventral periventricular nucleus (AVPe), medial preoptic area (MPA) and periventricular nucleus (Pe) A method for testing a substance that provides an excitatory stimulus, comprising:
providing pyroglutaminated RF amide peptide (QRFP)-producing neurons;
contacting the test compound with the cell;
measuring the excitation of the QRFP-producing neurons;
determining whether a test compound provides an excitatory stimulus to the QRFP-producing neurons by comparing excitation of the QRFP-producing neurons before and after contact with the test compound.
(10c) specifically to pyroglutaminated RF amide peptide (QRFP)-producing neurons located within the regions of the anterior ventral periventricular nucleus (AVPe), medial preoptic area (MPA) and periventricular nucleus (Pe) A method for testing a substance that provides an excitatory stimulus, comprising:
administering a test compound to the regions of the anterior ventral periventricular nucleus (AVPe), medial preoptic area (MPA) and periventricular nucleus (Pe) of a mammal;
measuring the excitation (e.g., potential) of QRFP-producing neurons;
determining whether a test compound provides an excitatory stimulus to the QRFP-producing neurons by comparing excitation of the QRFP-producing neurons before and after contact with the test compound.
(10d) A method of testing for a test compound that induces hibernation, comprising:
administering a test compound to regions of the anterior ventral periventricular nucleus (AVPe), medial preoptic area (MPA) and periventricular nucleus (Pe) of a mammal (e.g., a non-human mammal);
confirming that the mammal hibernates;
A method, including
(10e) The method according to (10d) above,
From the correlation between the deep body temperature (e.g., intestinal temperature) and the oxygen consumption of the mammal (e.g., non-human mammal), the deep body temperature (theoretical set temperature) when the oxygen consumption is 0 and ΔVO ₂ /ΔT _B (heat production feedback gain);
A method wherein both the theoretical setpoint temperature and the negative feedback gain of heat production are reduced by administration of the test compound relative to pre-administration, indicating that the mammal has hibernated.
(10f) A method of testing whether a test compound induces hibernation in a mammal, such as a human, comprising:
Estimates of the theoretical setpoint temperature and heat production feedback in humans in whom test compounds were administered to the regions of the anterior ventral periventricular nucleus (AVPe), medial preoptic area (MPA) and periventricular nucleus (Pe). providing an estimate of the gain and an estimate of the theoretical setpoint temperature of the human prior to administration and an estimate of the heat production feedback gain;
An estimated theoretical set temperature and an estimated feedback gain of heat production after administration of said test compound compared to an estimated theoretical set temperature and an estimated feedback gain of heat production prior to administration of said test compound. and checking whether both of
A decrease in the estimated theoretical setpoint temperature and the estimated feedback gain of heat production after administration than before administration indicates that the mammal has hibernated.
(10g) A method for determining (predicting, estimating, computationally calculating) whether a test compound induces hibernation in a mammal such as a human, comprising:
Estimates of the theoretical setpoint temperature and heat production feedback in humans in whom test compounds were administered to the regions of the anterior ventral periventricular nucleus (AVPe), medial preoptic area (MPA) and periventricular nucleus (Pe). providing an estimate of the gain and an estimate of the theoretical setpoint temperature of the human prior to administration and an estimate of the heat production feedback gain;
An estimated theoretical set temperature and an estimated feedback gain of heat production after administration of said test compound compared to an estimated theoretical set temperature and an estimated feedback gain of heat production prior to administration of said test compound. and checking whether both of
A decrease in the estimated theoretical setpoint temperature and the estimated feedback gain of heat production after administration than before administration indicates that the mammal has hibernated.
(10h) A method for determining (predicting, estimating, computationally calculating) whether a test compound induces hibernation in a mammal such as a human, comprising:
In a mammal, such as a human, to which the test compound was administered to the regions of the anterior ventral periventricular nucleus (AVPe), medial preoptic area (MPA) and periventricular nucleus (Pe), pre- and post-administration, respectively recording oxygen consumption and core body temperature under at least two different ambient temperature conditions;
estimating the correlation between oxygen consumption and core body temperature before and after administration, respectively;
Determining from the estimated correlation whether the degree of decrease in oxygen consumption when core body temperature is decreased is decreased after administration compared to before administration, and when oxygen consumption is 0 determining whether the estimate of core body temperature, given the
The degree of decrease in oxygen consumption when core body temperature decreases is lower after administration than before administration, and the estimated value of core body temperature when assuming that oxygen consumption is 0 is The method, wherein the decrease after administration compared to before indicates that said mammal has hibernated.
(11) A method for determining whether a test compound induces hibernation in mammals such as humans (examination, prediction, estimation, computational scientific calculation),
In a mammal, such as a human, to which the test compound was administered to the regions of the anterior ventral periventricular nucleus (AVPe), medial preoptic area (MPA) and periventricular nucleus (Pe), pre- and post-administration, respectively providing (or recording) recorded oxygen consumption and core body temperature under at least two different ambient temperature conditions, respectively, in
estimating the correlation between oxygen consumption and core body temperature before and after administration, respectively;
Determining from the estimated correlation whether the degree of decrease in oxygen consumption when core body temperature is decreased is decreased after administration compared to before administration, and when oxygen consumption is 0 determining whether the estimate of core body temperature, given the
The degree of decrease in oxygen consumption when core body temperature decreases is lower after administration than before administration, and the estimated value of core body temperature when assuming that oxygen consumption is 0 is The method, wherein the decrease after administration compared to before indicates that said mammal has hibernated.
(12) A device for determining hibernation,
In a mammal, such as a human, to which the test compound was administered to the regions of the anterior ventral periventricular nucleus (AVPe), medial preoptic area (MPA) and periventricular nucleus (Pe), pre- and post-administration, respectively a recording unit that records oxygen consumption and core body temperature recorded under at least two different ambient temperature conditions, respectively, in
Before and after administration, the correlation between oxygen consumption and core body temperature was estimated. and whether the estimated core body temperature, assuming zero oxygen consumption, is reduced post-dosing compared to pre-dosing. and a calculation unit for determining
The degree of decrease in oxygen consumption when core body temperature decreases is lower after administration than before administration, and the estimated value of core body temperature when assuming that oxygen consumption is 0 is and a determination unit that determines that the mammal has hibernated if it is decreased post-administration compared to pre-administration.

Figures 1a-h relate to activation of Qrfp-iCre neurons that lower hypothalamic body temperature and energy expenditure. FIG. 1a shows a strategy for chemogenetic excitation of iCre-positive neurons in Qrfp-iCre mice. Chemical excitation of iCre-positive cells in Qrfp-iCre mice was found to induce hypothermia as measured by infrared thermography. Heterozygous (Q-het) or homozygous (Q-homo) Qrfp-iCre mice carrying heterozygous Rosa26 ^dreaddm3 (M3) and/or Rosa26 ^dreaddm4 (M4) alleles were subjected to experiments. Distribution of Q neurons in Qrfp-iCre mice. Depicted by the expression of GFP after injection of AAV10-DIO-GFP into the medial basal hypothalamus. Scale bar (horizontal image), 500 μm; inset, 100 μm; coronal image, 200 μm. Pe: periventricular nucleus, AVPe: anterior chamber Pe, MPA: medial preoptic area, LPO: lateral preoptic area, AHA: anterior hypothalamus, VMH: ventromedial hypothalamus, LHA: lateral hypothalamus, SON: suprathalamus Nucleus, DMH: dorsomedial hypothalamus, TMN: tuberomaminal nucleus, MM: medial mammary nucleus, SCN: suprachiasmatic nucleus, VOLT: vascular organ of the lamina septum, TC: optic nucleus, ARC: optic vein, No. 3-ventricular optic nucleus. Representative thermometry results showing surface body temperature of Q-hM3D mice. CNO was injected intraperitoneally at 0 hours. Note the temperature rise in the tail at 0.5 hours (arrow). Fos immunostaining of slices from Q-hM3D mice 90 minutes after CNO IP. Scale bar, 100 μm. Procedure for metabolic analysis by chemogenetic activation of Q neurons in Q-hM3D mice. Chronological progression of hypothermia/hypometabolism after activation of Cre-positive neurons by DREADD. purple strain, Q-hM3D mice; yellow strain, Qrfp-iCre mice injected with AAV10-DIO-hM3Dq-mCherry into the lateral hypothalamus; black strain, Qrfp-iCre mice injected with AAV-DIO- into the medial basolateral hypothalamus; Inject mCherry (negative control). Q neuron-induced hypometabolism (QIH) persists for several days and can be induced again by CNO infusion. Lines and shading in b and g indicate the mean and standard deviation of each group, respectively. Figures 2a-l show the results of histological and functional analysis of Q neuron projections. FIG. 2a shows the strategy of injecting AAV-DIO-GFP into Qrfp-iCre mice to delineate the axonal projection pattern of Q neurons delineated by expressing GFP in Q neurons. AVPe, MPA and Pe. Distribution of GFP-positive Q neurons in scale bar, 100 μm. Distribution of axons arising from Q neurons. Scale bar, 100 μm. Cropped images of images taken with the brain using the ScaleS method were revealed by the ScaleS method, showing AVPe Q neurons and DMH fibers. In situ hybridization analysis showing that populations of Q neurons express Vgat and/or Vglut2 in Q-hM3D mice. Scale bar, 100 μm. High magnification image of the rectangular area shown in Fig. 2e. A single-color image of the rectangular area in Fig. 2e. Higher magnification images of rectangular areas 1-3 shown in FIG. 2f showing Q neurons expressing Vgat, Vglut2 or both. (1) Vgat ⁺ mCherry ⁺ ; (2) Vglt2 ⁺ mCherry ⁺ ; (3) Vgat ⁺ Vglt2 ⁺ mCherry ⁺ . Percentage of Vgat-positive neurons (1291 of 1997 cells), Vglut2 (359 of 1997 cells) and (115 of 1997 cells) in mCherry-expressing cells (counted in 4 sections prepared from 2 mice). Other mCherry-expressing cells express neither Vgat nor Vglut2. Strategies for optogenetic excitation of Q neurons or their axons in DMH and RPa, scale bar, 100 μm. Body temperature course measured by a thermographic camera during photogenic excitation in DMH or RPa of Cre-positive cells or their axons in AVPe/MPA. Four shots of light stimulation are indicated by blue arrowheads. Lines and shading indicate the mean and standard deviation for each group, respectively. The lower panel shows representative thermographic images obtained by excitation of Q neurons (AVPe/MPA). Note that the tail shows heat release 5 minutes after the first light stimulation (arrow). Estimated T _S 30 minutes after the fourth light stimulation. Note that the effect of DMH fiber stimulation on Ts is roughly equivalent to that of cell body excitation in AVPe/MPA. Pelvis, periventricular nucleus; AVPe, anterior chamber Pe; VOLT, vascular organ of the septum septum; MPA, medial preoptic area; VLPO, ventrolateral hypothalamic area; PVN, hypothalamic paraventricular nucleus; MM, medial papillary nucleus; LC, locus coeruleus; PAG, periaqueductal gray matter; LPB, lateral paranucleus; RVLM, paraventricular nucleus; 3 globus pallidus; nucleus pallidus; chamber. Q neuron-induced hypometabolism is accompanied by a reduction in body temperature set point. Evolution of _TB and _VO2 of _QIH at various TA. QIH was induced in Q-hM3D mice by CNO injection. Lines and shading indicate the mean and standard deviation for each group. Minimum T _B (left) and VO ₂ (right) under normal and QIH conditions. Schematic representation of heat production and loss pathways in mammals. Heat loss is proportional to the difference between T _A and T _B at the G factor. Thermogenesis is governed by the difference between T _R and T _A in factor H. Relationship between _{TB - TA} and _VO2 at various TA. The slope of the curve indicates G. Dots are recorded data, the thick line is drawn from the median posterior G and the thin line is the curve drawn from 500 Gs randomly selected from the posterior sample. Backward distribution of the estimated G(e) and the difference in G(f) from QIH to normal conditions. Backward distribution of the estimated G(e) and the difference in G(f) from QIH to normal conditions. Relationship between _TB and _VO2 at various _TA . The negative slope of the curve indicates H and the x-intercept indicates TR. See Figure 3d for an explanation of the dots and lines. Distribution of estimated H(h) and difference of H from QIH to normal state(i). Distribution of estimated H(h) and difference of H from QIH to normal state(i). Distribution of estimated TR (j) and difference in TR from QIH to normal (k). Distribution of estimated TR (j) and difference in TR from QIH to normal (k). Metabolic translocation of QIH within an individual. The upper row shows the transition of animal posture at various TAs during _QIH . The second column is the time expansion rate of the third column, each showing the metabolic transition of one representative animal. Note that mice exhibit a curled-up posture during FIT at T _A =28° C. (B) and an extended posture during QIH at T _A =28° C. (D). Even during _QIH when the TA was lowered to 12° C., as in FIT(E), the animals assumed a curled-up position, indicating that the animals were positioned to avoid heat loss. Figures 4a-g show that Q neurons play a role in inducing fasting-induced diapause in mice. Figure 4a shows a strategy for suppressing Q neuron function. Left panel, experimental procedure. Right panel, TeTxLC-eYFP expression in AVPe/MPA shown by immunostaining with anti-GFP antibody. Scale bar, 100 μm. Schematic of FIT experiment. Normal FIT was abolished by expressing TeTxLC in Q neurons. Note that these mice did not show a rapid oscillatory decline in metabolism. Minimum VO ₂ at 24-36 h and 36-48 h were compared between control and TeTxLC mice. Inhibition of Q neurons blocked the _VO2 reduction normally seen in FIT. Estimated differences in minimum VO2 between control and _TeTxLC mice were [0.01, 0.80] ml/g/h at 24-36 hours and [0.36, 1.16] at 36-48 hours. ml/g/h. Small SDs of _TB and VO2 in _TeTxLC mice indicate that Q neurons are involved in abrupt changes in metabolism, including oscillatory changes during FIT. The “>” and “<” signs indicate whether the estimated minimum difference 89% HPDI or the standard deviation from TeTxLC to control mice is negative or positive. FIT was induced in control, Qrfp-iCre heterozygous and homozygous mice, indicating that deletion of the QRFP peptide does not affect FIT. Procedure to delineate input neurons that make monosynaptic contacts with Q neurons using recombinant rabies virus vectors. Distribution of input neurons of the Q neuron. Arrows indicate starting cells. Scale bar, 100 μm. Brain regions containing input neurons. Scale bar, 100 μm. Pe, periventricular nucleus; AVPe, anterior ventricle Pe; MPA, medial preoptic area; VOLT, vascular organ of the septum; MnPO, median preoptic area; VMPO, ventromedial preoptic area; VLPO, ventral hypothalamus Field; PVN, hypothalamic paraventricular nucleus; TC, tuber; opto, optic; ac, anterior commissure; f, fornix; 3V, third ventricle. FIG. 5 shows a schematic of the apparatus of the first embodiment. FIG. 6 shows a schematic of the apparatus of the first embodiment. FIG. 7 shows a schematic of the apparatus of the second embodiment. FIG. 8 shows a schematic of additional configurations of the apparatus of the first and second embodiments.

Specific description of the invention

As used herein, "subject" means humans and non-human mammals, for example non-human primates such as rats, monkeys, gorillas, chimpanzees, orangutans and bonobos.

As used herein, "hypothalamus" is the center located in the diencephalon and responsible for the regulation of endocrine and autonomic functions. As used herein, "Q-neurons" refer to nerves located in the medial regions of the hypothalamus, namely the anterior ventral periventricular nucleus (AVPe), medial preoptic area (MPA) and periventricular nucleus (Pe) A neuronal cell that produces pyroglutaminated RF-amide peptide (QRFP). Pyroglutaminated RF-amide peptide (QRFP) is a neuropeptide identified as an endogenous ligand for the GPR103 receptor. QRFP is strongly expressed in the hypothalamus and is thought to be involved in the regulation of sleep and wakefulness, as it has been shown to have the effect of enhancing the wakefulness system.

As used herein, “T _A ” means ambient temperature of the subject (° C.), “T _B ” means core body temperature (° C.), and “T _R ” means theoretical setpoint temperature (° C.). do. " _VO2 " means oxygen consumption of a subject. T _R is the body temperature determined as T _B when VO ₂ is zero by determining the correlation between T _B and VO ₂ when T _A is varied. T _B is the internal body temperature, not the body surface temperature, which is affected by the outside air temperature. For example, _TB in humans can be defined by rectal, intraesophageal, intravesical, or intrapulmonary blood temperature. The heat generation negative feedback gain (H) indicates heat generation efficiency and is given by H=ΔVO ₂ /ΔT _B.

As used herein, "hibernation" is a state of hypothermia and low metabolism found in mammals. "Daily torpor" is a short-term state of low metabolism. Hibernation and circadian dormancy differ in that circadian _dormancy causes a decrease in H with little decrease in TR, whereas hibernation causes a significant decrease in both _TR and H. As used herein, "hibernation-like state" means a state in which both _TR and H are significantly decreased with decreasing _TA . As used herein, the term "non-hibernating animal" refers to an animal that does not have a habit of hibernating in winter or during fasting.

As used herein, "exhaled breath" is breath exhaled by a subject. As used herein, the oxygen concentration is an index indicating the amount of oxygen per volume. The units of oxygen concentration can be, for example, % or mmHg. As used herein, "oxygen consumption" ₍ VO2) is the oxygen consumption per hour calculated from the oxygen concentration contained in exhaled and inspired air. Since oxygen consumption varies with body weight, it may be corrected and calculated per unit body weight (eg, per kg and per g).

We found that in the brain of a living, non-hibernating animal subject, the hypothalamus consists of the anterior ventral periventricular nucleus (AVPe), the medial preoptic area (MPA) and the periventricular nucleus (Pe). It has been found that a hibernation-like state can be induced in the subject by applying an excitatory stimulus to pyroglutaminated RF amide peptide (QRFP)-producing neurons within one or more regions selected from the group.

Thus, according to the present invention, there is provided a method of inducing a hibernation-like state in a living non-hibernating animal subject, comprising: and periventricular nucleus (Pe).

Excitatory stimulation can be induced by stimulating with deep brain electrodes, stimulating with an activator of QRFP-producing neurons.

According to the present invention, in the brain of a living subject, one or more selected from the group consisting of the anterior ventricular periventricular nucleus (AVPe), the medial preoptic area (MPA) and the periventricular nucleus (Pe). A device (hereinafter sometimes referred to as the "device of the present invention") is provided for stimulating pyroglutaminated RF amide peptide (QRFP)-producing neurons in a region.
(A1) The device of the present invention is
a control unit that transmits a control signal for controlling voltage generation;
a voltage generator that receives a control signal from the controller and generates a voltage;
a stimulation probe electrically connected proximally to said voltage generator and having an electrical stimulation electrode distally, said voltage generator having a length sufficient to access QRFP-producing neurons from the brain surface; and a stimulation probe for generating electrical stimulation at a distal electrical stimulation electrode with voltage from the body. Thus, the device of the present invention can electrically provide excitatory stimulation to QRFP-producing neurons. Alternatively, from the viewpoint of giving chemical stimulation instead of electrical stimulation, (A2) the device of the present invention is
a control unit that transmits a control signal that controls the release of the QRFP-producing neuron-stimulating compound;
a reservoir of said compound;
and a compound release unit that receives a control signal from the control unit and releases the compound from the compound storage unit.
(B) the device of the present invention comprises:
outside temperature gauge and
a core thermometer and
an exhaled gas analyzer for measuring oxygen concentration in exhaled gas;
a recording unit that records the measured outside temperature and at least one numerical value selected from the group consisting of core body temperature and oxygen concentration;
may further include In the configuration (B) of the apparatus of the present invention, the subject can check whether the core body temperature (T _B ) decreases as the outside temperature (T _A ) decreases, and the exhaled gas analysis result , the theoretical set temperature (T _R ) and heat production negative feedback gain (H) can be determined. This allows the device of the present invention to determine whether or not the subject has induced a hibernation-like state.

The device of the present invention will be specifically described below.
(First embodiment)
In a first embodiment, the device of the present invention has the configuration (A1) above. The device of the present invention thereby induces a hibernation-like state in a subject by electrically stimulating QRFP-producing neurons in the brain of a living subject. In the following, a first embodiment is described with reference to FIGS. 5 and 6. FIG.

The device 1 of the present invention comprises:
a control unit 10 that transmits a control signal for controlling voltage generation;
a voltage generator 20 that receives a control signal from the controller and generates a voltage;
a stimulation probe electrically connected proximally to said voltage generator and having an electrical stimulation electrode distally, said voltage generator having a length sufficient to access QRFP-producing neurons from the brain surface; and a stimulation probe 30 for generating electrical stimulation at a distal electrical stimulation electrode 40 with voltage from the body.

In the device 1 of the present invention, the control section 10 transmits a control signal for controlling voltage generation. The control unit 10 may include control elements (microprocessor and power supply or battery). The control signals can control one or more voltage generations with one control signal. Alternatively, this control signal can be sent multiple times to control multiple voltage generations. The control signal can apply a first-order voltage stimulus, but may control voltage generation, for example, to apply multiple stimuli until a hibernation-like state is induced in the subject {however, hibernation A stimulus may or may not be added after induction of the like state}.

In the device 1 of the present invention, the voltage generator 20 is electrically connected to the controller 10 by the wiring 15, and can receive a control signal from the controller 10 and generate a voltage. The voltage can be, for example, a voltage between 0 and 5 volts (V), eg, 0.5 volts (V). It can be varied in l volts. The voltage can be pulsed, for example, with a pulse width of, for example, tens of microseconds, and a stimulation frequency of, for example, tens to hundreds of pps. The voltage may be adjusted, for example, starting at 1 volt and increasing until an effect is observed.

Although an example in which the control unit 10 and the voltage generation unit 20 are connected by the wiring 15 has been described, in the device 1 of the present invention, the control unit 10 and the voltage generation unit 20 are connected by As shown in FIG. 2, wireless communication may be enabled between the control signal transmitter 11 included in the controller 10 and the control signal receiver 21 included in the voltage generator. In this aspect, the voltage generator 20 can have a battery 20a. Battery 20a may be chargeable in a contactless manner. If the battery 20a can be charged in a non-contact manner, the battery 20a can be charged from the outside of the body even if it exists inside the body.

The voltage generator 20 transmits the voltage generated by the voltage generator 20 to the stimulation probe 30 and the stimulation electrode 40 present at the tip through the extension cable 25 . The distal (ie, tip) of the stimulation probe 30 has a stimulation electrode 40 that can apply a voltage to brain tissue.

The stimulation probe 30 can be inserted into the brain by stereotactic neurosurgery to precisely reach the QRFP-producing neurons with the stimulation electrodes 40 . In stereotaxic brain surgery, the head is fixed in a measurement frame, and electrodes are inserted into positions determined by CT scan or MRI with an accuracy of 1 mm or less. From the viewpoint of stereotaxic brain surgery, the stimulation probe 30 is made of a hard material (for example, a hard material such as tungsten) to the extent that it does not bend or stretch when puncturing deep into the brain. The stimulation probe 30 may have a diameter of, but not limited to, approximately 1 μm to 1 mm, or approximately 1 mm to 2.5 mm. Stimulation probe 30 has one or more (eg, two, three, or four) stimulation electrodes 40 distally. The stimulation electrode 40 can have a length of about 1-5 mm in the long axis direction of the stimulation probe 30 . When the stimulation probe 30 has a plurality of stimulation electrodes 40, the stimulation electrodes 40 can be arranged at intervals of about 1 mm to 1.5 mm, although not particularly limited. Each of the stimulation electrodes 40 may be collectively controlled by a single control signal, or preferably each may be separately controlled by individual control signals. By separately controlling each by an individual control signal, it is possible to selectively generate a voltage to the optimum electrode in relation to the insertion position of the electrode to stimulate the brain.

The device 1 of the present invention induces a hibernation-like state in a subject and need not be portable. Portable here means moving with the object relative to the footing where the object is located (eg, the ground, or the floor of the vehicle if in a vehicle). Thus, the device of the invention can be fixed in place. Since the device of the invention can be connected to a power source, it can, for example, have no batteries or rechargeable batteries.

(Second embodiment)
While the first embodiment disclosed a device for electrical stimulation of the deep brain, the second embodiment relates to a device for chemical stimulation of the deep brain. A second embodiment will be described below with reference to FIG.

In a second embodiment, the device 100 of the invention comprises
a control unit 110 that transmits a control signal that controls the release of the QRFP-producing neuron-stimulating compound;
a reservoir 125 of said compound;
a compound delivery unit 120 that receives a control signal from the control unit and delivers the compound from a compound storage unit 125;
a guide 130 comprising a compound outlet 140 and a channel for the compound to the outlet 140 and delivering the compound to QRFP-producing neurons;
have In the device 100 of the present invention, the control section 110 is electrically connected to the compound delivery section 120 through the wiring 115 . Compound delivery unit 120 receives a control signal from control unit 110, and in response to the control signal, releases the compound accumulated in storage unit 125 from storage unit 125 through channel 126, channel 121, and guide 130 to the compound outlet. Released from 140 into the brain. The compound may be in the form of a solution dissolved in a solvent, and may be delivered to the compound outlet 140 by the liquid delivery mechanism of the compound delivery section 120 . The compound storage unit 125 may have a compound introduction port 125a for introducing a compound from the outside. Compound inlet 125a can supply compound to the compound reservoir. Compound reservoir 125 may be exposed to the outside of the body. However, when compound reservoir 125 is exposed to the outside of the body, compound reservoir 125 is maintained under sterile conditions. The control unit 110 transmits a control signal to the compound delivery unit 120 so that, for example, 1 μL to 100 μL of liquid is delivered per compound delivery.

The guide 130 can be inserted into the brain by stereotactic neurosurgery to direct the compound outlet 140 precisely to the QRFP-producing neurons. In stereotaxic brain surgery, the head is fixed in a measurement frame, and electrodes are inserted into positions determined by CT scan or MRI with an accuracy of 1 mm or less. From the viewpoint of stereotactic brain surgery, the guide 130 is made of a hard material (for example, a hard material such as tungsten) that does not bend or stretch when the guide is punctured into the deep part of the brain. Stimulation probe 30 may, for example, have a diameter on the order of 1 mm to 2.5 mm.

The device 100 of the present invention induces a hibernation-like state in a subject and need not be portable. Portable here means moving with the object relative to the footing where the object is located (eg, the ground, or the floor of the vehicle if in a vehicle). Thus, the device of the present invention may be fixed to the installation site (eg, the bed on which the subject lies or the floor on which the bed is arranged). Since the device of the invention can be connected to a power source, it can, for example, have no batteries or rechargeable batteries.

(additional configuration)
The device 1 of the first embodiment and the device 100 of the second embodiment have the configuration (B):
an outside temperature gauge 50;
a thermometer 60;
an exhaled gas analyzer 70 for measuring oxygen concentration in exhaled gas;
It may further have a recording unit 80 that records the measured outside air temperature and at least one numerical value selected from the group consisting of body temperature and oxygen concentration {here, the thermometer preferably measures the core body temperature of the subject. It can be a core thermometer that measures}. The above (B) may be provided in the control unit 10 or the control unit 110, for example, as shown in FIG. Each of them is connected to the voltage generator 20 by wire or wirelessly as described in the first embodiment and the second embodiment}. In inducing a hibernation-like state in a subject, the ambient temperature (or ambient temperature of the subject) (T _A ) is lowered, as well as the subject's core body temperature (T _B ) and metabolism. Accordingly, by providing an ambient temperature gauge for measuring the ambient temperature (or the target's ambient temperature) and a thermometer (preferably, a core thermometer), the device of the present invention can measure the target's body temperature (preferably, the core temperature) It becomes possible to monitor the relationship with temperature.

In addition, the apparatus of the present invention can estimate the oxygen consumption ₍ VO ₂ ) by the subject by providing the breath gas analyzer 70 for measuring the oxygen concentration in the breath gas. can be used to estimate the metabolic state of a subject.

Also, from core body temperature (T _B ) and oxygen consumption (VO ₂ ), it is possible to estimate the theoretical set temperature of body temperature (T _R ) and feedback gain of heat production (H). The theoretical setpoint temperature of body temperature (T _R ) is the variation of core body temperature (T _B ) and oxygen consumption (VO ₂ ) while changing (e.g., decreasing) the ambient temperature (or subject's ambient temperature) (T _A ). A relationship is determined and determined as an estimate of core body temperature (T _B ) when oxygen consumption (VO ₂ ) is zero. The relationship between core body temperature (T _B ) and oxygen consumption (VO ₂ ) can be determined, for example, by linear regression. Also, the heat generation feedback gain (H) can be determined as H=ΔVO ₂ /ΔT _B.

The device of the present invention may further include a recording unit 80 that records the measured outside air temperature and at least one numerical value selected from the group consisting of body temperature (preferably core body temperature) and oxygen concentration. The apparatus of the present invention may further comprise an oxygen consumption determiner 90 for determining the subject's oxygen consumption from the oxygen concentration in the exhaled gas. The apparatus of the present invention may further comprise an estimator 91 for estimating the theoretical set temperature of body temperature (T _R ) and the feedback gain of heat production (H). The apparatus of the present invention may further comprise a determination unit 92 for determining whether the subject has induced a hibernation-like state from the theoretical set temperature of body temperature (T _R ) and the feedback gain of heat production (H). The device of the present invention may further comprise an output 93 of information as to whether a hibernation-like state has been induced. Examples of the output unit 93 include a display that displays the information and/or a printer that prints the information. Information as to whether or not the hibernation-like state has been induced includes information that the hibernation-like state has been induced and information that the hibernation-like state has not been induced, and can be output by the output unit 93 .

(Third embodiment)
According to the invention,
A device for determining hibernation, comprising:
In a mammal, such as a human, to which the test compound was administered to the regions of the anterior ventral periventricular nucleus (AVPe), medial preoptic area (MPA) and periventricular nucleus (Pe), before and after administration, respectively a recorder for recording oxygen consumption (VO2) and core body temperature ( _TB ) recorded under at least _two different ambient temperature (T _A ) conditions, respectively, at
Before and after administration, the correlation between oxygen consumption and core body temperature was estimated. and whether the estimate of core body temperature, assuming zero oxygen consumption, is reduced post-dosing compared to pre-dosing. and a computing unit that determines
When the core body temperature decreases, the degree of decrease in oxygen consumption decreases after administration compared to before administration, and the estimated value of core body temperature when the oxygen consumption is assumed to be 0 is and a determination unit that determines that the mammal has hibernated if it is decreased after administration compared to before.

The recorder records oxygen consumption (VO ₂ ) and core body temperature (T _B ) recorded under at least two different ambient ambient temperature (T _A ) conditions. The recording unit associates and stores one VO ₂ and one _TB with one _TA . The recorded oxygen consumption (VO ₂ ) and core body temperature (T _B ) are read from the recording unit and transmitted to the computing unit, where the correlation between oxygen consumption and core body temperature is estimated. . In one aspect, the correlation is linear. After the correlation is estimated, the computing unit determines whether the degree of decrease in oxygen consumption when core body temperature is decreased is decreased after administration compared to before administration, and Determine whether the estimate of core body temperature, assuming zero consumption, is lower after dosing compared to before dosing. Based on the determination by the calculation unit, the determination unit determines that the degree of decrease in oxygen consumption when the core body temperature decreases is lower after administration than before administration, and the oxygen consumption is 0. It can be determined that the mammal has hibernated when the hypothetical estimate of core body temperature is reduced after administration compared to before administration. The determination unit determines whether the degree of decrease in oxygen consumption when the core body temperature decreases does not decrease after administration compared to before administration, or the core body temperature when the oxygen consumption is assumed to be 0. If the estimated value does not decrease after administration compared to before administration, it can be determined that the mammal has not hibernated (or it can be determined that it has not hibernated).

The device for determining hibernation in the third embodiment of the present invention may further comprise a deep body thermometer and an expired gas analyzer for measuring oxygen concentration in expired gas. The device according to the third embodiment may further include an output unit that receives information on determination regarding hibernation from the determination unit and outputs the information. The information output unit can be a user interface such as a display, can be a recording device to a non-volatile memory such as a USB memory and an SD card, can be an information transmission device for wireless communication to the outside, or can be a printer It can be a printing device for media such as paper.

The apparatus of the first embodiment or the second embodiment may further include the apparatus for determining hibernation of the third embodiment.

(Stimulation method of the present invention)
According to the present invention, a method is provided for reducing the theoretical set point of body temperature and/or the feedback gain of heat production in a subject. According to the invention, a method of inducing a hibernation-like state in a subject is provided.
According to the method of the invention, it comprises providing an excitatory stimulus to pyroglutaminated RF amide peptide (QRFP)-producing neurons. The present invention also provides a method of reducing the feedback gain of heat production in a subject comprising providing an excitatory stimulus to pyroglutaminated RF amide peptide (QRFP)-producing neurons. Also in accordance with the present invention is a method of reducing the theoretical set point of body temperature and the feedback gain of heat production in a subject, comprising providing an excitatory stimulus to pyroglutaminated RF amide peptide (QRFP)-producing neurons. , a method is provided. The present invention also provides a method of inducing a hibernation-like state in a subject, the method comprising providing an excitatory stimulus to pyroglutaminated RF amide peptide (QRFP)-producing neurons using a drug or the like. be.

In the methods of the invention, pyroglutaminated RF-amide peptide (QRFP)-producing neurons can be stimulated, for example, using the device of the invention. In the method of the present invention, the QRFP-producing neurons can be stimulated by applying a voltage to the QRFP-producing neurons. In the method of the present invention, a receptor (e.g., hM3Dq) is expressed specifically using pyroglutaminated RF amide peptide (QRFP)-producing neurons, for example, using the DREADD method, and a ligand for the receptor (e.g., clozapine QRFP-producing neurons can be stimulated by administering -N-oxide (CNO). hM3Dq can be expressed in QRFP-producing neurons by infecting the QRFP-producing neurons of a subject with a virus (e.g., adenovirus, adeno-associated virus, etc.) having a gene encoding hM3Dq operably linked to the QRFP promoter. . CNO can be administered to the brain, for example, by the device of the invention.

In the methods of the invention, pyroglutaminated RF amide peptide (QRFP)-producing neurons can also be stimulated with activators of these neurons. Activators can be screened using QRFP neurons or searched using cultured cells in which receptors expressed in QRFP neurons are forced to express. Neuronal activators may be administered locally to QRFP-producing neurons using an applicator. QRFP-producing neuron-specific activators may be administered by systemic administration, such as intracerebroventricular and intrathecal and intravenous administration.

The method of the invention may further comprise reducing the ambient temperature. This can reduce the _TB of the target. It is thought that a decrease in _TB in a hibernation-like state leads to a hypometabolism state, which makes it possible to maintain life by reducing energy consumption.

The methods of the invention may further comprise measuring the core body temperature (T _B ) of the subject. The method of the present invention may further comprise measuring oxygen concentration in exhaled breath of the subject.

Methods of the invention may further comprise estimating the subject's oxygen consumption (VO ₂ ). A subject's oxygen consumption (VO ₂ ) can be estimated, for example, from the difference in inspired and expired oxygen concentrations.

The method of the present invention is a method of administering a test compound in a mammal, such as a human, to the regions of the anterior ventral periventricular nucleus (AVPe), the medial preoptic area (MPA) and the periventricular nucleus (Pe). providing (or recording) recorded oxygen consumption and core body temperature under at least two different ambient temperature conditions before and after administration, respectively;
estimating the correlation between oxygen consumption and core body temperature before and after administration, respectively;
Determining from the estimated correlation whether the degree of decrease in oxygen consumption when core body temperature is decreased is decreased after administration compared to before administration, and when oxygen consumption is 0 determining whether the estimate of core body temperature, assuming that the
When the core body temperature decreases, the degree of decrease in oxygen consumption decreases after administration compared to before administration, and the estimated value of core body temperature when the oxygen consumption is assumed to be 0 is A decrease after administration compared to before can be a method of indicating that said mammal has hibernated.
In this aspect, the method of the invention may further comprise estimating a theoretical set point (T _R ) for the subject's body temperature. The theoretical setpoint temperature (T _R ) is the relationship between core body temperature (T _B ) and oxygen consumption (VO ₂ ) while varying (e.g., decreasing) the ambient temperature (or subject's ambient temperature) (T _A ). and taken as an estimate of core body temperature (T _B ) when oxygen consumption (VO ₂ ) is zero. The relationship between core body temperature (T _B ) and oxygen consumption (VO ₂ ) can be determined, for example, by linear regression.

The method of the present invention may further comprise estimating the subject's heat production feedback gain (H). The heat generation feedback gain (H) can be determined as H=ΔVO ₂ /ΔT _B.

Methods of the invention may further comprise determining whether the subject is in a hibernation-like state. Whether or not a subject is in a hibernation-like state can be determined by whether both the theoretical setpoint temperature of body temperature (T _R ) and the feedback gain of heat production (H) decrease when the ambient temperature is decreased. can. A subject can be determined to be in a hibernation-like state if both the theoretical setpoint temperature of body temperature (T _R ) and the feedback gain of heat production (H) decrease when the ambient temperature is decreased.
A hibernation-like state can be beneficial in improving life-preserving functions by slowing down the body's metabolism.

(Screening system of the present invention)
According to the present invention, pyroglutaminated RF amide peptide (QRFP)-producing neurons present in the regions of the anterior ventral periventricular nucleus (AVPe), medial preoptic area (MPA) and periventricular nucleus (Pe). A method for screening a substance that provides an excitatory stimulus, comprising:
contacting the isolated QRFP-producing neurons with a test compound;
measuring the excitation of the QRFP-producing neurons;
selecting a test compound that provides an excitatory stimulus to the QRFP-producing neurons;
A method is provided, comprising: The method can be an in vitro method.

The excitation of QRFP-producing neurons can be measured electrically. Electrical measurement of neuronal excitation can be performed, for example, by an electrophysiological technique using a conventional method, using membrane potential depolarization as an indicator. Membrane potential can be measured, for example, by a nerve recording method such as a microelectrode method or a patch clamp method, or may be measured using a fluorescent probe for measuring membrane potential. The fluorescent probe for membrane potential measurement is not particularly limited, but 4-(4-(didecylamino)styryl)-N-methylpyridinium iodide (4-Di-10-ASP), bis-(1,3-dibutylbarbi trimethine oxonol turic acid (DiSBAC2(3)), 3,3′-dipropylthiadicarbocyanine iodide (DiSC3(5)), 5,5′,6,6′-tetrachloro-1,1′ , 3,3′,-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) and rhodamine 123. Neuronal excitation can also be measured chemically. Intracellular calcium concentration is increased.For example, neuronal excitation can be measured using a calcium concentration indicator.As a calcium concentration indicator, 1-[6-amino-2-(5-carboxy-2-oxazolyl )-5-benzofuranyloxy]-2-(2-amino-5-methylphenoxy)ethane-N,N,N′,N′-tetraacetic acid, pentaacetoxymethyl ester (Fura 2-AM) and various Probes are known and can be used in the present invention.

QRFP-producing neurons are neurons present in the regions of the anterior ventral periventricular nucleus (AVPe), medial preoptic area (MPA), and periventricular nucleus (Pe), and can be regarded as established neurons. can. As the established neuron strain, a strain obtained by selecting a strain in which neurons produce QRFP can be used. Whether a neuron produces QRFP can be confirmed by a conventional method using an antibody against QRFP.

(Method for judging hibernation of the present invention)
The method for determining hibernation of the present invention analyzes the effect of a drug that induces, is expected to induce, or has the potential to induce hibernation in a subject. If the subject enters a hibernation-like state, it can be maintained or released. If the subject does not enter hibernation, further treatment can be given or treatment can be discontinued.
The hibernation determination method of the present invention can be a computational scientific method. The hibernation determination method of the present invention may not include medical procedures.

The method for determining hibernation of the present invention comprises:
A method for determining whether or not a test compound induces hibernation in a mammal such as a human or the possibility thereof (examination, prediction, estimation, computer scientific determination), comprising:
In a mammal, such as a human, to which the test compound was administered to the regions of the anterior ventral periventricular nucleus (AVPe), medial preoptic area (MPA) and periventricular nucleus (Pe), before and after administration, respectively providing (or recording) recorded oxygen consumption and core body temperature under at least two different ambient temperature conditions, respectively, in
estimating the correlation between oxygen consumption and core body temperature before and after administration, respectively;
Determining from the estimated correlation whether the degree of decrease in oxygen consumption when core body temperature is decreased is decreased after administration compared to before administration, and when oxygen consumption is 0 determining whether the estimate of core body temperature, given the
When the core body temperature decreases, the degree of decrease in oxygen consumption decreases after administration compared to before administration, and the estimated value of core body temperature when the oxygen consumption is assumed to be 0 is A decrease after administration compared to before can be a method of indicating that said mammal has hibernated. A mammal can be a non-human mammal.

Setting of different ambient environmental temperature conditions can be performed by the temperature control unit (for example, the apparatus of the first embodiment or the second embodiment). Oxygen consumption and core body temperature can be determined by a breath gas analyzer and core thermometer, respectively. As the breath gas analyzer and core thermometer, those provided in the device of the first embodiment or the second embodiment can be used.

[Experimental method]
(1) Animals All animal experiments were performed in accordance with animal experiment guidelines at the International Institute of Sleep Medicine (IIIS), University of Tsukuba, and RIKEN Biosystems Dynamics Research Center (BDR). NIH guidelines were followed as approval was obtained from the respective institutional Animal Care and Use Committees. Mice were fed and watered ad libitum, except for diapause induction experiments, and were maintained at a T _A of 22° C., 50% relative humidity, and a 12 h light/12 h dark cycle. Mice weighing >34 g were found not to exhibit reproducible FIT, so heavy mice >34 g were excluded from diapause experiments.

Qrfp-iCre mice were generated by homologous recombination in C57BL/6N embryonic stem cells and transplantation in 8-cell stage embryos (ICR). A targeting vector was designed to replace the entire coding region of the prepro-Qrfp sequence in exon 2 of the Qrfp gene with the iCre and pgk-Neo cassettes so that the endogenous Qrfp promoter drives iCre expression. Chimeric mice were bred to C57BL/6J females (Jackson Labs). The pgk-Neo cassette was deleted by mating FLP66 mice that were backcrossed to C57BL/6J mice at least 10 times. First, F1 hybrids from mating heterozygotes with heterozygotes were made. These mice were backcrossed to C57BL/6J mice at least 8 times.

Rosa26 ^dreaddm3 and Rosa26 ^dreaddm4 mice were generated by homologous recombination in C57BL/6N embryonic stem cells, followed by the same procedure as in Qrfp-iCre mice above.

(2) Virus AAV was produced using a triple transfection, ^helper -free method as previously described33. The final purified virus was stored at -80°C. Recombinant AAV vector titers were determined by quantitative PCR. AAV ₁₀ -EF1α-DIO-TVA-mCherry, 4×10 ¹³ ; AAV ₁₀ -CAG-DIO-RG, 1×10 ¹³ ; AAV ₁₀ -EF1α-DIO-hM3Dq-mCherry, 1.64× ₁₀ ¹² ; AAV _2/9 -hsyn-DIO-TeTxLC-GFP, 6.24 x ₁₀ ¹⁴ ; AAV _2/9 - mCherry, ^1.44 x 10 ¹² ; hsyn-DIO-GFP, 4×10 ¹² genome copies/ml. Recombinant rabies vectors have been generated by previously reported methods ^22,34 . The titer of SADΔG-GFP(EnvA) was 4.2×10 ⁸ infectious units/ml.

(3) Surgery For injection of AAV vectors, male Qrfp-iCre heterozygous mice (8-12 weeks old) were anesthetized with isoflurane and placed in a stereotaxic frame (David Kopf Instruments).

For chemical genetic manipulation, Qrfp-iCre mice were injected with AAV ₁₀ -EF1α-DIO-hM3Dq-mCherry at a rate of 0.1 μm/min using a Hamilton needle and hypothalamic (for MB injection, anteroposterior ( medial lateral (ML), ±0.25 mm; dorsoventral (DV), −5.75 mm; 0.50 μl each site; LH injection; AP, −1.00 mm; ±1.00 mm; DV, −5.00 mm; 0.30 μl each site). The needle was held for 10 minutes after injection.

For optogenetic manipulation, AVPe (AP, 0.38 mm; ML, 0.25 mm; DV, −5.50 mm from bregma) was unilaterally injected with AAV10-EF1α-DIO-SSFO-EYFP. Then, on both sides above the AVPe (AP: 0.38 mm, ML: ±0.25 mm, DV: -5.20 mm) and on both sides of the DMH (AP: -1.70 mm, ML: ±0.25 mm, DV: −4.75 mm) or on one side of RPa (AP: −6.00 mm, ML: 0.00 mm, DV: −5.50 mm) (Fig. 2j). After a recovery period of at least 2 weeks in individual cages post-injection, mice were subjected to infrared thermal imaging experiments. Behavioral data were included only when these viruses were specifically targeted to Q neurons and the fiber optic implants were precisely placed.

(4) Recording biological signals For thermographic analysis, mice were placed in experimental cages (25 x 15 x 10 cm) and an infrared thermal imaging camera (InfReC R500EX; NIPPON AVIONICS) placed 30 cm above the cage floor. monitored using One day before the start of the experiment, the dorsal hair was removed with a shear for clear detection of the surface temperature. Thermograms of DREADD and light generation experiments were collected at 0.5 Hz and 1 Hz, respectively, and analyzed with the InfReC Analyzer NS9500 professional software (NIPPON AVIONICS). The maximum temperature of one frame was used as the animal's _TS (Fig. 1d).

Each animal was housed in a temperature-controlled chamber (HC-100, Shin Factory or LP-400P-AR, Nihon Ika Kikai Seisakusho Co., Ltd.) to record core body temperature, oxygen consumption, EEG, ECG, and respiratory patterns. For continuous recording of T _B (intraperitoneal temperature), a telemetry temperature sensor (TA11TA-F10, DSI) was implanted in the peritoneal cavity of animals under general inhalation anesthesia at least 7 days prior to recording. Animals' VO2 and carbon dioxide excretion rate ₍ VCO2) _were recorded continuously with a respiratory gas analyzer (ARCO-2000 mass spectrometer, ARCO Systems). _The respiratory index was calculated from the _VCO2 /VO2 ratio.

EEG and ECG were recorded by an implantable telemetry transmitter (F20-EET or HD-X02, DSI). For EEG recording, two stainless steel screws (1 mm diameter) were soldered to the wires of the telemetry transmitter and cranial cortex (AP, 1.00 mm; right, 1.50 mm from bregma or lambda) under general anesthesia. ). Two other wires from the transmitter were placed on the surface of the thoracic cavity and the ECG was recorded. Allow at least 10 days to recover from surgery. The EEG/ECG data acquisition system consisted of a transmitter, an analog-to-digital converter, and a recording computer with software Ponemah Physiology Platform (version 6.30, DSI). The sampling rate was 500 Hz for both EEG and ECG and the data were converted to ASCII format for review. Heart rate was evaluated by visual observation of waveforms.

Respiratory flow was recorded by a non-invasive respiratory flow recording system ³⁵ . Specifically, mice were placed in a metabolic chamber (TMC-1213-PMMA, Minamiderika Shokai) with an airflow of at least 0.3 L/min. The chamber was connected to a pressure sensor (PMD-8203-3G, Biotex) to detect the pressure difference between the outside and inside of the chamber. When an animal is breathing, the outside-to-inside pressure difference is greater during inspiration and less during expiration ³⁵ . The analog signal output from the sensor was digitized at 250 Hz by an AD converter (NI-9205, National Instruments) and stored in a computer by data logging software developed by Biotex.

(5) FIT Induction Induction experiments of circadian dormancy (torpor) were designed to record the animal's metabolism for at least 3 days. Animals were introduced into the chambers the day before the start of recording (day 0). Food and water were available ad libitum. _TA was set as indicated on day 0 and kept constant throughout the experiment. The telemetry temperature sensor implanted in the mouse was turned on before entering the chamber. The standard experimental design was as follows. On day 2, ZT-0, food was removed to induce diurnal torpor. After 24 hours, on day 3, each animal was returned to food at ZT-0.

(6) Recording metabolism during drug administration The DREADD agonist CNO (clozapine N-oxide, Abcam, ab141704) was dissolved in saline at a dose of 100 μg/mL and frozen at -20°C. The CNO solution was thawed on site and mice were administered the solution intraperitoneally at a dose of 1 mg/kg. CHA (N ⁶ -cyclohexyladenosine, Sigma-Aldrich, C9901), an adenosine _A1 receptor agonist, was dissolved in saline at a concentration of 250 μg/mL and intraperitoneally administered to mice at a dose of 2.5 mg/kg.

(7) Metabolic recordings during general anesthesia In addition to the T _B , VO ₂ and video recordings described above (see 'Recording of biological signals'), the entrance of the metabolic chamber was the exit of the inhalation anesthesia machine (NARCOBIT-E). , Natsume Seisakusho Co., Ltd.). 1% isoflurane was given at T _A =28° C. for 30 minutes followed by T _A =12° C. for 90 minutes. After the experiment, the animals were warmed on a hotplate and checked for recovery.

(8) Immunohistochemical staining Mice were deeply anesthetized with isoflurane and transcardially perfused with 10% sucrose in water, followed by ice-cold 4% paraformaldehyde in 0.1 M phosphate buffer pH 7.4. (4% PFA) and the brain was removed. Brains were post-fixed in 4% PFA overnight at 4°C, incubated in 0.1 M phosphate-buffered saline pH 7.4 (PBS) in 30% sucrose overnight at 4°C, and placed in a cryomold. of Tissue-Tek O.; C. T. They were immersed in compound (Sakura) and frozen at −80° C. until sectioning. Using a cryostat (CM1860, Leica), cells were coronal sliced into 4 equal series every 50 μm, collected in a 6-well plate filled with ice-cold PBS, and washed 3 times with PBS at room temperature (RT). The following incubation steps were performed with gentle shaking on an orbital shaker unless otherwise noted. Brain slices were incubated in 1% Triton X-100 in PBS for 1 hour at room temperature. Sections were blocked with 10% Blocking One (NACALAI TESQUE) in 0.3% Triton X-100-treated PBS (blocking solution) for 1 hour at room temperature without shaking. Sections were incubated overnight at 4°C in primary antibody diluted in blocking solution (diluent and type of each antibody listed below), then washed three times and incubated with secondary antibody overnight at 4°C. , PBS, then mounted and coverslipped with HardSet Antifade Mounting Medium containing DAPI (VECTASHIELD).

The initial antibodies used in this study were rabbit anti-cFos (1:4000, ABE457, Millipore), goat anti-mCherry (1:15000, AB0040-200, SICGEN), rat anti-GFP (1:5000, 04404-84, NACALAI TESQUE), mouse anti-TH (1:1000, sc-25269, Santa Cruz Biotechnology), mouse anti-orexin A (1:200, sc-80263, Santa Cruz Biotechnology), and rabbit anti-MCH (1:2000, M8440, SIGMA). Secondary antibodies are as follows. Alexa Fluor 488 donkey anti-rat, 488 donkey anti-rabbit, 594 donkey anti-rabbit, 594 donkey anti-goat, 647 donkey anti-mouse, and 647 donkey anti-rabbit (1:1000, Invitrogen). For Nissl staining, sections were counterstained with NeuroTrace 435/455 Blue Flue Fluorescent Nissl Stain (1:500, N-21479, Invitrogen) during the secondary antibody step and coverslips were blotted with FluorSave Reagent (Millipore). covered. Brain regions were determined using mouse brain maps by ^Paxinos and Franklin36.

(9) In situ hybridization Fluorescence in situ hybridization is performed using RNAscope Fluorescent Multiplex Kit (Advanced Cell Diagnostics), using a probe designed for RNAscope Fluorescent Multiplex in situ hybridization (ACDBio RNAscope 6-M3-Mr4Prob4 #1 #1 , mCherry#43201, Mm-Slc32a1#319191, Mm-Slc17a6#319171). Brains were dissected, immediately frozen in 2-methylbutane on dry ice, and stored in freezing embedding medium at -80°C. Brains were cooled to -16°C in a cryostat for 1 hour prior to sectioning. Brains were cut coronally into 20 μm sections using a cryostat (Leica CM1860UV) and mounted on Superfrost Plus Microscope slides (Fisherbrand). The pretreatment method and RNAscope Fluorescent Multiplex Assay were performed exactly according to the RNAsope Assay Guide (document numbers 320513 and 320293, respectively).

(10) Retrograde tracking of Q neurons Male Qrfp-iCre mice (10-12 weeks old) were injected with the following viruses. AAV ₁₀ -DIO-TVA-mCherry and AAV ₁₀ -DIO-RG were delivered to express TVA-mCherry and RG in Q neurons in the MB region (see above for procedures and coordinates). Two weeks later, SADΔG-GFP(EnvA) was injected at the same site. A Leica TCS SP8 laser confocal microscope and a Zeiss Axio Zoom. V16 was used to detect starter and input (single GFP-positive) neurons in whole brain sections, respectively.

(11) Blood Chemistry Test Blood was collected from anesthetized mice by left ventricular puncture using a 25 gauge needle. The collected blood was not stored on ice for more than 2 hours. Samples were centrifuged at 2,000G for 10 minutes at 4°C and the supernatant was collected and frozen at -30°C. Frozen serum samples were sent to FUJIFILM Wako Pure Chemical Corporation and analyzed for Na (mEq/L), K (mEq/L), Cl (mEq/L), AST (IU/L), ALT (IU/L), LDH ( IU/L), CK (IU/L), GLU (mg/dL) and total ketone body (μmol/L) concentrations were measured.

(12) Electrophysiological analysis Mice were decapitated under deep anesthesia with isoflurane (Pfizer). Brains were extracted and chilled in ice-cold cutting solution containing (mM): 125 mM choline chloride, 25 mM _NaHCO3 , 10 mM D(+)-glucose, 7 mM MgCl2, _2.5 mM KCl. , 1.25 mM _NaH2PO4 , and 0.5 mM CaCl2 _bubbled with _O2 (95%) and _CO2 ( ₅ %). Horizontal brain slices (250 μm thick) containing the hypothalamus were prepared with a vibratome (VT1200S, Leica) and maintained for 1 hour at room temperature in artificial CSF (ACSF) containing (mM): 125 mM NaCl, 26 mM NaHCO ₃ , 10 mM D(+)-glucose, 2.5 mM KCl, 2 mM CaCl ₂ , 1 mM MgSO ₄ bubbled with O ₂ (95%) and CO ₂ (5%). Electrodes (5-8 MΩ) were filled with an internal solution containing (in mM): 125 mM K-gluconate, 10 mM HEPES, 10 mM phosphocreatine, 0.05 mM tolbutamide, 4 mM NaCl. , 4 mM ATP, ₂ mM MgCl2, 0.4 mM GTP, and 0.2 mM EGTA, pH 7.3, adjusted with KOH). Firing of hM3Dq-mCherry expressing neurons was recorded in current-clamp mode at a temperature of 30°C. CNO (1 μM) was applied in bath and the effect was investigated. A combination of a MultiClamp 700B amplifier, Digidata 1440A A/D converter and Clampex 10.3 software (Molecular Devices) was used to control membrane voltage and data acquisition.

(13) 3D Imaging of Clear Mouse Brain Clear mouse brains were prepared by the ScaleS method as previously described ³⁷ . Urea crystals (Wako Pure Chemical Industries, 217-00615), D(-)-sorbitol (Wako Pure Chemical Industries, 199-14731), methyl-β-cyclodextrin (Tokyo Chemical Industry, M1356), γ-cyclodextrin (Japanese Ko Pure Chemical Industries, 037-10643), N-Acetyl-L-Hydroxyproline (Skin Essential Actives, Taiwan), Dimethylsulfoxide (DMSO) (Wako Pure Chemical Industries, 043-07216), Glycerol (Sigma, G9012) and Triton X -100 (Nacalai Tesque, 35501-15) was used to make the scale solution. Brains of Qrfp-iCre mice injected with AAV-DIO-GFP were fixed and cleared with ScaleS. Images were obtained with a laser confocal microscope (Olympus, XLSLPN25XGMP (NA 1.00, WD: 8 mm) (RI: 1.41-1.52)).

(14) Statistical Analysis In this study, we applied Bayesian statistics to evaluate our hypotheses and experimental results. We designed a statistical model with parameters representing the hypothetical structure and fitted the model to the experimental results. Bayesian inference estimates the posterior probability distribution of model parameters from the likelihood distribution and prior probability distribution of the parameters. The posterior distribution provides information about how the model can explain the hypothesis from the experimental results. A Bayesian model can explicitly include all types of uncertainty, so it can handle data about noise in the observations, or it can handle a few of samples can be fully utilized. In addition, it uses a hierarchical model and can handle multiple layers of multiple groups with different numbers of samples. All of these advantages of Bayesian inference make it well-suited to address problems commonly encountered in animal experiments. Model fitting was performed using Hamiltonian Monte Carlo with non-U-turn sampler, adaptive variant, as run in version 2.18.0 of Stan with RStan library ³⁸ in version ^3.5239 of R. did. inspecting trace plots,

and estimated convergence by estimating the number of valid samples. The prior probability density function of the model is defined with weak informationality and conservativeness and is specified in the following sections. The basic principles and techniques for designing statistical models are based on the book Statistical ^Rethinking40 . Both the model and the data source code used for the analysis are available at https://briefcase. liken. Available from jp/public/JjtgwAnqQ81AgyI. (Will be protected with password 'qih' and made public for evaluation).

Body weight of Qrfp-iCre mice was modeled by a state-space hierarchical model (code folder QRFP_KO_BW) at a given age and strain. Animals in each group; wild-type (n = 9), heterozygous (n = 9), and homozygous (n = 10) Qrfp-iCre mice were housed in each cage without individual identification. Unobservable baseline body weight was defined as the time variable B _t,s , where t is the time point and strain index (1, 2, and 3 for wild-type, heterozygous, and homozygous Qrfp-iCre mice, respectively). with a trend η _t , _s and a total time T, the observed state Y _t,i can be described by modeling the observation error with a lognormal distribution as follows:

A uniform prior probability density function was applied to all parameters except σ1 and σ2, which are drawn from standard half-normal distributions.

The spike frequency of Qrfp-positive neurons in brain slices was modeled by parameterizing the difference in spike frequency when neurons were activated by CNO (code folder Patch_M3_CNO). If the total number of slices is K and the observed spike frequencies of control and CNO-dosing recordings in the i-th slice are B _i and C _i , respectively, then B _i is modeled by β _BASE with observational error and C _i is modeled by the sum of β _BASE and β _CNO with observation error. Since the spiking frequency is a positive real number, the error can be modeled by a lognormal distribution, so B _i and C _i can be written as

All σ were sampled from a standard semi-normal distribution.

The _TS of photostimulated animals was modeled with a hierarchical multi-layer model (Fig. 21, code folder SSFO_Opto). Four groups of animals were included in this experiment. _TS was recorded at 1 Hz and median values were saved every 10 seconds for further analysis. All T _s recorded 115-125 minutes after the first photostimulation were included in the analysis. Where K is the total number of animals and Y is the _TS during the time of interest for mouse j belonging to i, Y is the global mean parameter _β , the group parameter β _GROUP , and the individual mouse parameter β _MOUSE .

All σ were sampled from a standard semi-normal distribution. Between-group differences in _TS were compared by estimating the mean _TS of each group from the posterior distribution, which is the sum of β and β _GROUP with normally distributed noise at the standard deviation of σ _MOUSE .

To assess the thermoregulatory system under QIH and normal conditions, animal heat loss and heat production were described in a hierarchical multilayer model (Fig. 3c-k, code folder QIH_GTRH). Three parameters G, TR and H in two metabolic conditions, normal and _QIH , were estimated from the metabolically stable state of animals at different _TA . The detailed method was previously described ³ . In short, a linear model consisting of controllable and observable parameters _TB and VO2 was _fitted to experimental results for _{both TB} and _VO2 , using _TA as a predictor with normally distributed noise. let me The posterior distributions of the slope and intercept coefficients of each model were then used to estimate G, T _R , and H. In this analysis, the prior probability density function of the standard deviation of the noise has a standard semi-normal distribution, and the other parameters have a uniform distribution except for the intercept coefficient of _TB using a uniform distribution due to negative values. used the positive area of .

Circadian changes in metabolism in Q-TeTxLC mice were analyzed by modeling metabolism by clustering recorded values into L and D phases (code folder TeTxLC_LD). In particular, if Y is the observed _TB of group i in phase j, then Y can be expressed as the sum of the differences between basal metabolism (L-phase metabolism) and D-phase, with a normally distributed observation noise of become.

All σ were sampled from a standard semi-normal distribution. _In modeling VO2, the basic model structure was identical to _TB modeling, except that observation errors were modeled as log-normal distributions, since VO2 assumes _only positive real numbers.
Metabolism during FIT in Q-TeTxLC mice was modeled with a hierarchical multilayer model (Fig. 4d, code folder TeTxLC_FIT). The minimum value Y for a group i in section j can be expressed as the sum of the group β _0[i] 's mean metabolism and the difference parameter β _1[i,j] .

Mouse identity was included as a predictor of observed Y values to model metabolic variance. In this way, Y for a given group of a SECTION is modeled as a normal distribution, which averages the mouse-dependent mean α _MOUSE and the group- and section-dependent parameters β _{GROUP,SECTION} as: σ _{GROUP, SECTION} was used as standard deviation.

All σ in equations (22)-(24) and (28)-(30) were sampled from a standard semi-normal distribution. These models were used to model T _B , VO ₂ and RQ. Even Y in these models could theoretically accept negative real numbers, and the latter converged well, so we also applied this model to _VO2 and RQ.

[Experiment and results]

Example 1: Induction of hypometabolism by a chemically defined population of hypothalamic neurons The hypothalamic neuropeptide pyroglutaminated RF-amide peptide (QRFP) was originally aimed at discovering new RF-amide peptides. discovered through bioinformatics approaches ^9,10 . A Qrfp peptide was also identified and purified from rat brain as an endogenous ligand for the orphan G-protein coupled receptor hGPR103 ¹¹ . The prepro-Qrfp mRNA is localized only to the hypothalamus and is distributed in the periventricular nucleus (Pe), lateral hypothalamic area (LHA), and ridge gray (TC) ¹¹ . Qrfp has been implicated in food intake, sympathetic modulation, and anxiety ^11,12 . The inventors generated mice in which codon-improving Cre recombinase (iCre) was knocked into the Qrfp gene (Qrfp-iCre mice). We inserted CAG-hM3Dq-mCherry into the Rosa26 locus with an upstream floxed transcription stop element to obtain mice expressing hM3Dq-mCherry only in iCre-expressing neurons (Qrfp-iCre; Rosa26 ^dreaddm3 mice). Crossed with Rosa26 ^ddreadm3 mice. During excitatory chemogenetic experiments with Qrfp-iCre;Rosa26 ^dreaddm3 mice, these mice exhibited markedly reduced motor activity and, ultimately, intraperitoneal (IP) administration of clozapine-N-oxide (CNO). Severe and persistent immobility began approximately 30 minutes after injection. We noticed that the posture of these mice was similar to that observed during tropor, suggesting that activation of iCre-positive cells in Qrfp-iCre mice induced a tropor-like state. , was first hypothesized to be characterized by immobility and low _TB (as discussed below, the hypothermia induced here has been shown to be a hibernation-like state rather than diurnal dormancy). To test this hypothesis, we measured surface body temperature (T _S ) using a thermographic camera and found that CNO-induced immobility in Qrfp-iCre;Rosa26 ^dreaddm3 mice was accompanied by marked and persistent hypothermia. (Fig. 1b). The decrease in _TS began approximately 5 minutes after CNO administration and persisted for approximately 12 hours. Mice then spontaneously recovered from hypothermia without external rewarming.

In contrast, inhibitory DREADD manipulation via activation of hM4Di in iCre-positive neurons of Qrfp-iCre;Rosa26 ^dreaddm4 mice did not show any effect on T _S (Fig. 1b). Importantly, hM3Dq-mediated activation of iCre-positive neurons in Qrfp-iCre;Rosa26 ^dreadm3 mice was suppressed even in homozygous Qrfp-iCre mice in which the prepro-Qrfp sequences were completely replaced by iCre in both alleles. It induced severe hypothermia (Fig. 1b). This suggests that the Qrfp peptide itself is not essential for inducing hypothermia. Rather, the extent of hypothermia was more pronounced in Qrfp knockout (Qrfp-iCre homozygous) mice, suggesting that endogenous Qrfp itself may counteract hypothermia. This is consistent with our previous observations11 that ^Qrfp increases sympathetic outflow and increases heart rate and blood pressure upon central administration.

Therefore, Qrfp was identified as a chemical marker of hypothermia-induced neurons. Since iCre-positive neurons are only observed in the hypothalamus but are distributed in several discrete hypothalamic regions in Qrfp-iCre mice, we next attempted to identify hypothermic-inducing hypothalamic regions. Cre-activating AAV vectors with flip-excision (FLEX) switch ¹³ were injected into the thalamus of Qrfp-iCre mice using two different stereotaxic coordinates; medial-basal (MB) or lateral (LH) injection (see Methods). iCre-positive neurons in the lateral and medial regions of the hypothalamus were separately manipulated by injecting into the hypothalamus. MB injection of Cre-dependent AAV vectors can express specific genes in iCre-positive neurons in the medial regions of the hypothalamus: the anterior ventral periventricular nucleus (AVPe), medial preoptic area (MPA) and Pe. It could, but could not be expressed in LHA (Fig. 1c). Multicolor fluorescence in situ hybridization analysis confirmed that the majority of mCherry-positive cells in these regions expressed Qrfp mRNA. After MB injection of Qrfp-iCre mice with AAV ₁₀ -EF1a-DIO-hM3Dq-mCherry to express hM3Dq in this region, electrophysiological studies were performed using hypothalamic slices made from these mice, We confirmed that bath application of CNO strongly excited mCherry-positive neurons. IP injection of CNO into these mice was found to cause deeper and longer lasting hypothermia than the severe immobility observed in Qrfp-iCre;Rosa26 ^dreaddm3 mice (Fig. 1b, 1d). A very low _TB state (below 30 °C) persisted for more than 48 h (Fig. 1d). Immunostaining with anti-Fos and anti-mCherry antibodies revealed numerous mCherry and Fos double-positive neurons in AVPe, MPA and Pe, confirming the in vivo excitation of these neurons by CNO (Fig. 1e).

From these observations, we concluded that iCre-positive neurons in the AVPe/MPA and Pe of Qrfp-iCre mice (these neurons are referred to below as quiescence-induced neurons or Q neurons) are primarily responsible for the induced hypothermic state. In the following experiments, we used Qrfp-iCre mice with MB injection of AAV ₁₀ -EF1a-DIO-hM3Dq-mCherry (referred to as Q-hM3D mice) for induction of hypothermia, unless otherwise stated. was basically used.

To further analyze the induced hypothermic state, Q-hM3D mice were implanted with telemetric temperature sensors intraperitoneally and metabolism was continuously analyzed by respiratory gas analysis (Fig. 1f). The present study demonstrated that CNO-induced hypothermia in Q-hM3D mice was accompanied by a marked decrease in the rate of _O2 consumption (VO2:oxygen consumption) ( _Fig . 1g), with a concomitant decrease in _TB with _TS after CNO administration. Confirmed to do. In contrast, excitatory DREADD manipulation of iCre-positive neurons in the lateral hypothalamic area (LHA and TC) of Qrfp-iCre mice by LH injection of AAV ₁₀ -EF1a-DIO-hM3Dq-mCherry did not induce hypothermia. (Fig. 1g).

During the Q neuron-induced hypothermia/hypometabolism (QIH) state, heart rate was markedly decreased (758 beats/min and 215 beats/min, 2 h before and 2 h after CNO injection, respectively) (n=3). average). Respiratory rate decreased from 333 breaths/min to undetectable (tidal volume below detection limit). _At these timings, VO2 decreased from 3.60 to 1.17 ml/g/hr. During QIH, mice exhibited very low amplitude electroencephalograms (EEGs), distinct from those observed in non-rapid eye movement sleep characterized by high amplitude slow waves. Serum chemistry data suggest that blood glucose levels drop during QIH, presumably due to reduced gluconeogenesis due to reduced sympathetic tone. These observations further suggest that many physical functions are robustly reduced with decreases in _TB and VO2 during _QIH .

Although DREADD-mediated effects usually last only a few hours after CNO injection, DREADD-induced QIH in Q-hM3D mice persisted much longer. Surprisingly, at T _A =20° C., the QIH of T _B below 30° C. persisted for more than 48 h after a single dose of CNO (1 mg/kg), with complete return of VO ₂ to normal. It took about a week to complete (Fig. 1h). After recovery from QIH, mice were healthy and appeared to behave normally. QIH was reproducible after repeated CNO injections in the same mice, indicating the reversibility of this manipulation (Fig. 1h).

Example 2: Q neurons act on the dorsomedial hypothalamus to induce QIH To clarify the mechanism of QIH induction, the axonal projection of Q neurons was analyzed. After Qrfp-iCre mice were injected with AAV ₁₀ -EF1a-DIO-GFP to express GFP specifically in Q neurons (Fig. 2a,b), MPA, VOLT, paraventricular nucleus (PVN), supraoptic Nucleus (SON), dorsomedial hypothalamus (DMH), LHA, tuberomaminal nucleus (TMN), medial mammary nucleus (MM), periaqueductal gray matter (PAG), lateral parabrachial nucleus (LPB), locus coeruleus We observed GFP-positive fibers in the nucleus (LLC), rostral ventrolateral medulla (RVLM), and nucleus pallidum (RPa), a region involved in thermoregulatory regulation and sympathetic control (Fig. 2c) ¹⁴ . The inventors found that DMH received a particularly abundant projection. Analysis of the brain revealed by the ScaleS method further suggested the location of Q neurons and projections to the DMH (Fig. 2d).
Next, triple-color in situ hybridization of Q neurons was used to confirm whether these Q neurons were inhibitory or excitatory. After confirming that CNO injection effectively induces QIH in Q-hM3D mice, these mice were subjected to in situ hybridization histochemistry. Probes encoding transcripts encoding the excitatory and inhibitory markers mCherry, vesicular glutamate transporter 2 (Vglut2) and vesicular GABA transporter (Vgat) were used. We found that approximately 2/3 of Q neurons were Vgat positive and approximately 2/5 were Vglut2 positive (Fig. 2e-i).

Among the regions containing abundant projections by Q neurons (Fig. 2c), we focused on the DMH. This is because thermogenic neurons were previously identified in DMH ¹⁵ . An optogenetic approach was used to clarify the function of Q-neuron axonal projections to the DMH. Injection of AAV ₁₀ -DIO-SSFO-eYFP into Qrfp-iCre mice (Q-SSFO mice) resulted in expression of stabilized step function opsin (SSFO) ¹⁶ in Q neurons (Fig. 2j). SSFO was confirmed to be expressed in AVPe, MPA and Pe. To confirm the effect of optogenetic excitation on _TS , we first implanted an optical fiber into AVPe/MPA, where many somata of Q neurons are found (Fig. ) to induce photogenic excitation of SSFO-positive cell bodies in mice. In this state, optogenetic excitation of Q neurons rapidly induced strong hypothermia lasting approximately 20 min (Fig. 2k). Repeated excitation of Q neurons every 30 min for four times resulted in profound hypothermia with T _S as low as T _A (22° C.). Many Fos-positive neurons were identified in SSFO-eYFP-positive cells of AVPe/MPA after excitation (Fig. 2j). Optogenetic-evoked QIH was apparently shorter-lasting than QIH induced by hM3Dq-mediated pharmacogenetic excitation of Q neurons (Fig. 2k), indicating changes in gene expression profiles. suggest that Gq-mediated metabotropic signaling in Q neurons leading to Q may play a role in creating long-term persistence of QIH.

Q-SSFO mice were then bilaterally implanted with optical fibers in the DMH and light stimulation was applied to the axonal fibers. This manipulation effectively reduced _TS , but slightly weaker than that induced by somatic stimulation of AVPe/MPA (Fig. 2k, l). As a control, we also examined the effects of light stimulation of Q neuron fibers in RPa, as ^RPa is known to contain sympathetic premotor neurons for heat production via brown adipose tissue regulation. We also observed subtle effects of photogenetic excitation of Q neuron fibers in RPa on _TS (Fig. 2k,l). From these results, we hypothesized that Q neurons act primarily on DMH and to a lesser extent on RPa, inducing QIH.

Example 3: Theoretical Set Temperature Decreases During QIH An increase in temperature in the mouse tail was observed immediately after QIH induction and was induced by optogenetic or pharmacogenetic excitation of Q neurons, suggesting that _TB It was suggested that peripheral vessels dilate and release heat during depletion (Fig. 1d, Fig. 2k). A telangiectasia without an increase in T _B suggests a resetting of the theoretical body temperature set point (T _R ) below normal, as seen in the hibernating state of hibernating animals. To assess this, a characterization of the mouse thermoregulatory system during QIH was performed. Thermal conductance _{( G} ), H and TR can be estimated from T _B and VO2 under multiple ambient temperatures (T _A ) under conditions where the animal has no external work and metabolism is stable. Can ³ . Q-hM3D mice were prepared and T _B and VO ₂ were recorded during QIH under different T _A (8, 12, 16, 20, 24, 28 and 32° C.) (FIG. 3a). We compared the ₁₁ h mean _TB and VO2 after IP injection of saline or CNO. During _QIH , animals showed _{lower TB} and VO2 at all TAs compared to matched controls (Fig. 3b). When the heat production system is functioning properly, i.e., when TR is higher than _TB and the _{thermoregulatory} system is increasing _VO2 to reach TR ( _Fig . 3c), increasing _{TA TB} increases and _VO2 decreases. T _B and VO ₂ each exhibited a minimum at different T _A , with only coordinated heat production properties in the range of 16–24 ° _C (Fig. 3b). Therefore, metabolic data for T _A =16, 20, and 24° C. during QIH were used for further analysis. First, G was estimated from the relationship between T _B −T _A and VO ₂ (Fig. 3d). The 89% highest posterior density interval (HPDI) of G was [0.212, 0.221] ml/g/hr/°C and [0.182, 0.220] ml/g under normal and QIH conditions, respectively. /hr/°C (Fig. 3e; below 89% HPDI is shown in square brackets with two numbers). Quantitatively, the posterior distribution (ΔG) of the difference between both Gs was [−0.0040, 0.0348] ml/g/hr/°C (Fig. 3f), including 0, between normal conditions and QIH. This suggests that G under the condition is indistinguishable. This was in contrast to diurnal dormancy (torpor), which exhibits a lower G than normal conditions ³ . Second, we estimated H and TR from _TB and _VO2 ( _Fig . 3g). H was [3.43, 8.72] ml/g/hr/°C in normal state and [0.181, 0.369] ml/g/hr/°C in QIH (Fig. 3h), and each median was a decrease of 95.3%. The posterior distribution of the difference (ΔH) was [3.17, 8.48] ml/g/hr/°C (Fig. 3i) and was positive, suggesting that the probability of these conditions being different is 89% or greater. was done. This H-drop was similar to that during fasting-induced diapause (FIT) ³ . especially,
TR was estimated to be [36.04, _36.60 ] °C in normal conditions and [26.83, 29.13] °C in QIH (Fig. 3j). The difference in T _R was 8.41° C. at each median and the rear distribution of the difference (ΔT _R ) was [7.18, 9.57]° C., clearly demonstrating the drop in T _R during QIH. (Fig. 3k). Given the very small theoretical setpoint shift of ³ in FIT, this observation highlights the similarities between QIH and hibernation, as well as the differences between QIH and diurnal torpor.

To provide further evidence for TR reduction during _QIH , a hallmark of hibernation, we observed the relationship between posture and metabolism within individual mice when _TA was dynamically altered during QIH. (n=4, 1 representative data in Fig. 3l and 3 other data). The highly stable and prolonged hypometabolic state of QIH as in hibernating animals allowed it to be examined in mice. Q-hM3D mice were set at T _A =28° C. to induce FIT (FIG. 3l A and B). After 24 hours of recovery, QIH was induced by CNO administration (Fig. 3l C, D, E, and F). Interestingly, at T _A =28° C., mice exhibited an extended posture during QIH, which is normally seen in animals exposed to high temperature environments (FIG. 3lD). This was clearly different from the typical sitting posture observed during FIT at T _A =28° C. (FIG. 3lB). This behavioral observation further indicates that TR is lower in _QIH than in FIT and normal conditions. Furthermore, when the T _A was lowered to 12° C., the mice returned to a sitting position resembling circadian dormancy (torpor) (E in FIG. 3l) and shivering began. These results strongly support that during _QIH , TR declines, but physical function and behavior are still modulated to accommodate changes in _TA .

It is well known that animals have a low metabolic rate during hibernation, but their metabolism is actively regulated in response to _TA . Similarly, in _QIH , animals exposed to TAs below 16 °C exhibited significantly greater VO2 compared with animals exposed to TAs at 20 ° _C or 28 °C ( _Fig . 3b). ^In fact, this is similar to previous _reports18 of hibernating animals that showed increased metabolism when TA was lowered to a certain level. This active hypometabolism feature of QIH was also confirmed in individual animals (Fig. 3l). The behavioral and metabolic responses of mice during QIH were quite different from those observed under normal conditions where body functions attempt to maintain _TB within a narrow range.

To compare metabolic function during QIH to the anesthetic state, we recorded metabolic transitions during general anesthesia under multiple _TA . As expected, anesthetized animals showed no increase in _VO2 or change in posture when exposed to low _TA . We also investigated the metabolic state induced by systemic delivery of the adenosine A1AR agonist (6) N-cyclohexyladenosine (CHA), which has been used to induce hypothermia ¹⁹ . Although IP injection of CHA (2.5 mg/kg) into wild-type mice effectively induced hypothermia/ _{hypometabolic} state, mice were induced to have low T by either increased VO2 or behavior (posture and shivering). _A (12° C.) did not react. TB at 20°C tended to be higher in CHA-induced hypothermia _than in _QIH , while _TB and VO2 were further decreased in CHA-induced hypothermia. On the other hand, these parameters increased with QIH when TA was set at 12 ° _C (Fig. 3b). These observations indicate that QIH is completely different from general anesthesia or CHA-induced passive hypothermia, which induces a hypothermic state by blocking the regulatory system of _TB .

Example 4: Q neurons are involved in normal fasting-induced circadian dormancy (torpor). Because of this possibility, we investigated whether Q neurons are also involved in circadian dormancy (torpor). A common or similar mechanism may also play a role in inducing hibernation and diurnal dormancy (torpor) ²⁰ . To investigate the role of Q neurons in circadian dormancy (torpor), Qrfp-iCre mice (Q-TeTxLC mice) were injected with AAV _2/9 -hSyn-DIO-TeTxLC-eYFP to tetanus toxin specifically in Q neurons. We expressed the light chain (TeTxLC) and examined whether blockade of SNARE complex-mediated neurotransmission in Q neurons affected FIT (Fig. 4b). Co-injection of AAV2 _/ 9-hSyn-DIO-TeTxLC-eYFP and AAV10 _- EF1a-DIO-hM3Dq-mCherry completely abolished the effects of CNO on _TB , suggesting that blockade of the SNARE complex induces QIH in Q neurons. suggesting that the ability is lost. We found that the normal structure of FIT was disrupted in all Q-TeTxLC mice. No rapid oscillatory changes in metabolism were seen in these fasted mice (Fig. 4c,d). This suggests that Q neuron function is required to induce a rapid drop in _TB during FIT. Interestingly, the gradual decline in _TB observed in these mice implies the existence of a Q neuron-independent hypometabolic mechanism during FIT. In addition, Q-TeTxLC mice had less circadian variation in _TB than control mice, suggesting a major role for Q neurons in circadian regulation of _TB . Notably, homozygous Qrfp-iCre mice lacking the QRFP peptide showed normal FIT (Fig. 4e). These observations suggest that Q neurons, but not QRFP, are essential components in inducing circadian torpor and play an important role in the rapid shift in body temperature during circadian torpor. do.

To elucidate the neuronal mechanisms regulating the activity of Q neurons, we investigated the upstream of direct synaptic contact with Q neurons by recombinant pseudorabies virus vector (SADΔG(EnvA))-mediated labeling ²¹ (Fig. 4f). Neuronal populations were identified. After expression of TVA-mCherry and rabies glycoprotein (RG) in Q neurons using Cre-activated AAV vector ²² in Qrfp-iCre mice, SADΔG-GFP (EnvA) was injected at the same site. Starter cells that were double positive for TVA-mCherry and GFP were found in AVPe/MPA and Pe (Fig. 4g). Input neurons with direct synaptic input to Q neurons (positive for GFP but negative for mCherry) were identified in the median preoptic nucleus (MnPO), PVN and MPA (Fig. 4h). Input neurons were also observed in and around AVPe/Pe, suggesting the existence of local interneurons that modulate the function of Q neurons and the possibility that Q neurons form microcircuits with interneurons within AVPe/MPA and Pe. suggested. These observations suggest that Q neurons receive relatively sparse direct input from the intrahypothalamic region. Since FIT is induced by fasting, Q neurons are expected to monitor negative energy balance. Since PVH neurons have been shown to receive abundant inputs from the ARC ²³ , inputs from PVH to Q neurons may play a role in conveying information about trophic status. PVH input may also convey circadian information from the suprachiasmatic nucleus (SCN).

Since MPA is involved in the regulation of _TB ^24,25 , the mutual interaction between Q neurons and MPA may play an important role in thermoregulation. VMPO also contains input neurons. Previous studies identified temperature-sensitive neurons in the ventromedial preoptic nucleus (VMPO) as BDNF and PACAP double-positive neurons. Excitability of these cells also induced hypothermia ²⁶ . Although this effect is much smaller than that induced by Q neuron excitation, there may be a functional interaction of VMPO ^PACAP/BDNF neurons and Q neurons. It was also shown that DREADD excitation of TRPM2-positive cells in POA induces hypothermia. Since TRPM2 is ubiquitously highly expressed in the POA, including the AVPe/MPA and areas containing input neurons to Q neurons, TRPM2-induced hypothermia is induced by direct and/or indirect activation of Q neurons. ²⁷

Because Q neurons are localized along the third ventricle (3V) and the dendrites of these neurons extend along the ependymal and periventricular organ-proximal regions of the 3V (Fig. 1c), tanisites and ependymal Humoral factors released by cells, factors in cerebrospinal fluid, or capillaries may also be sensed.

[Discussion]
Here we show the existence of a novel hypothalamic neuron population with specific chemical (=Qrfp-expressing) and histological (AVPe/MPA) characteristics in mice, and the excitation of this population is highly similar to hibernation. Induce hypometabolism. This state, QIH, shares two main properties with hibernation. One is reduced TR and the other is actively regulated _{hypometabolism} . During QIH, mice actively regulate their bodily functions according to the external environment. A number of other physiological parameters such as slowed heart rate, weak breathing, and low voltage electroencephalograms suggest similarities between QIH and hibernation ²⁹ . Fos expression analysis showed in a previous study that cells around 3V were activated during hibernation in S. centilis ³⁰ . This activation pattern is very similar to the region where Q neurons are localized, suggesting that hibernating neurons may also use Q neurons to induce hibernation.

It is very surprising that mice were able to enter a hibernation-like state (=QIH). Because distantly related mammals, including rodents, canines, and even primates, have the ability to hibernate, the neural mechanisms of hibernation are conserved in a wide range of mammalian species, but these systems are absent in non-hibernating animals. It is reasonable to assume that under normal conditions they are not recruited. Since the Qrfp gene is also conserved in humans, it is speculated that excitation of Q neurons may exhibit an active hypometabolic state. In this study, we also confirmed that DMH is the major effector site of Q neurons. Future studies identifying QIH-inducing neurons in DMH will further clarify the mechanism of QIH. Q neurons may also act on other regions identified in this study. For example, SON was recently reported to play an important role in general anesthesia and sleep ³² .

We also found that Q neurons are required for fasting-induced circadian dormancy (torpor) in mice. However, by Fos staining or fiber photometry (data not shown), we were unable to repeatably detect an increase in Q-neuron activity during fasting in mice, suggesting that low-level activation of Q-neurons is associated with diapause (torpor). suggested that it might be sufficient to induce hypothermia.

Induced hibernation in non-hibernating animals demonstrated in this study is a promising step forward for understanding the neuronal mechanisms of active hypometabolism and how different tissues adopt a hibernation-like hypometabolic state. provide a method for Furthermore, with the future development of methods to selectively excite Q neurons, QIH will be of great interest in medicine with the potential to reduce systemic tissue damage after a heart attack or stroke, or useful for organ transplant preservation. It would provide a new approach for the development of methods that would enable the clinical application of synthetic hibernation in humans, which is an advantage.

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Claims (10)

Pyroglutamylation in one or more regions selected from the group consisting of the anterior ventral periventricular nucleus (AVPe), the medial preoptic area (MPA) and the periventricular nucleus (Pe) in the brain of a living subject. A device for stimulating RF amide peptide (QRFP)-producing neurons, comprising:
a control unit that transmits a control signal for controlling voltage generation;
a voltage generator that receives a control signal from the controller and generates a voltage;
a stimulation probe electrically connected proximally to said voltage generator and having an electrical stimulation electrode distally, said voltage generator having a length sufficient to access QRFP-producing neurons from the brain surface; a stimulation probe that generates electrical stimulation at a distal electrical stimulation electrode with a voltage from the body;
apparatus, including Pyroglutamylation in one or more regions selected from the group consisting of the anterior ventral periventricular nucleus (AVPe), the medial preoptic area (MPA) and the periventricular nucleus (Pe) in the brain of a living subject. A device for stimulating RF amide peptide (QRFP)-producing neurons, comprising:
a control unit that transmits a control signal that controls the release of the QRFP-producing neuron-stimulating compound;
a reservoir of said compound;
a compound delivery unit that receives a control signal from the control unit and delivers the compound from a compound storage unit;
a guide comprising a compound outlet and a channel for the compound to the outlet and delivering the compound to QRFP-producing neurons;
apparatus, including outside temperature gauge and
a core thermometer and
an exhaled gas analyzer for measuring oxygen concentration in exhaled gas;
3. The device according to claim 1 or 2, further comprising a recording unit for recording the measured ambient temperature and at least one numerical value selected from the group consisting of core body temperature and oxygen concentration. The recording unit records at least an outside temperature and a core body temperature,
4. The apparatus of claim 3, further comprising a determination unit that determines whether the subject is hypothermic from the ambient temperature and core body temperature recorded in the recording unit. The recording unit records at least an outside temperature, a core body temperature and an oxygen concentration,
4. The apparatus of claim 3, further comprising a determination unit that determines whether the subject is in a hypometabolism state from the ambient temperature, core body temperature, and oxygen concentration recorded in the recording unit. The recording unit records at least an outside temperature, a core body temperature and an oxygen concentration,
4. The apparatus according to claim 3, further comprising a determination unit that determines whether the subject is in a hibernation-like state from the ambient temperature, core body temperature, and oxygen concentration recorded in the recording unit. The control unit continuously or intermittently activates QRFP-producing neurons until it is determined that the subject is in any one state selected from the group consisting of a hypothermic state, a hypometabolic state, and a hibernation-like state. A device according to any one of claims 4 to 6, which transmits a control signal to stimulate. A method of lowering the theoretical setpoint temperature of body temperature in a non-human mammalian subject, the method comprising providing an excitatory stimulus to pyroglutaminated RF amide peptide (QRFP)-producing neurons. 8. The QRFP-producing neurons are neurons of one or more regions selected from the group consisting of the anterior ventral periventricular nucleus (AVPe), the medial preoptic area (MPA) and the periventricular nucleus (Pe). The method described in . 10. The method of claim 8 or 9, wherein the excitatory stimulus is a stimulus selected from the group consisting of chemical, magnetic and electrical stimulation.

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